CELL CYCLE AND GLUCOCORTICOID RECEPTOR PHOSPHORYLATION

细胞周期和糖皮质激素受体磷酸化

• 批准号: 2146835
• 负责人: ALLAN U MUNCK
• 金额: $ 16.86万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146835
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; apoptosis; cell cycle; corticosteroid receptors; flow cytometry; glucocorticoids; growth inhibitors; high performance liquid chromatography; hormone regulation /control mechanism; hydroxyurea; immunoprecipitation; mutant; phosphopeptides; phosphorus; phosphorylation; protein purification; protein sequence; radionuclides; receptor binding; reporter genes; sulfur; synchronous cell division; western blottings

Glucocorticoids at high doses are used widely to treat disorders ranging from minor allergies to leukemias and lymphoma. They act on almost all cells. So much effort has gone into maximizing their therapeutic effectiveness and minimizing unwanted side effects. Sensitivity to glucocorticoids varies strikingly through the cell cycle, the hormones being effective only if present during late G1 and S (DNA synthesis) phases. Our long-term goal is to elucidate the basic mechanisms of this variation. In this proposal, the central objective is to test the hypothesis that phosphorylation of glucocorticoid receptors (GRs) changes through the cell cycle and accounts at least in part for the variation in sensitivity. This hypothesis, based on evidence for cell cycle-dependence of GR number, nuclear binding, and phosphorylation, has gained support -- and the means to test it -- from our finding of a rapid, hormone-dependent increase in GR phosphorylation, and our identification by phosphopeptide mapping and sequencing of seven phosphorylated sites in mouse GRs: four lie in consensus sequences for the p34cdc2 kinases that control the cell cycle. The proposal is designed to answer the following specific questions; 1. Do hormone dependent GR hyperphosphorylation and/or GR phosphorylated sites change through the cell cycle? (a) Synchronized WCL2 cells (CHO cells with overexpressed mouse GRs) will be labeled with 32P and treated with hormone to assay hormone-dependent phosphorylation of the GRs; (b) Phosphorylated sites in these GRs will be identified by phosphopeptide mapping, and sequencing of any new peptides. 2. How do glucocorticoid sensitivity and GR number change through the cell cycle in Cho cells? Synchronized CHO cells with overexpressed GRs will be used to: (a) measure hormone effects on activity of a stably transfected gene and thymidine kinase; (b) determine if the increased number of GR binding sites in G1/S is due to increased GR protein per cell. 3. Do GR phosphorylation mutants behave differently from wild-type GRs? CHO cells with overexpressed GRs mutated at normally phosphorylated sites will be studied as in (2a). 4. Are GR phosphorylation and glucocorticoid-induced apoptosis in lymphocytes cell cycle-dependent, and modifiable by chemotherapeutic agents? Synchronized WEHI-7 cells will be used to measure (a) GR phosphorylation as in la; (b) glucocorticoid-induced apoptosis; (c) effects in (a) and (b) of cell cycle-modifying agents. Our results may benefit therapy and enhance understanding of glucocorticoid resistance.

高剂量的糖皮质激素广泛用于治疗各种疾病， 从轻微过敏到白血病和淋巴瘤。 他们几乎对所有 细胞 为了最大化他们的治疗效果， 有效性和最大限度地减少不必要的副作用。 对糖皮质激素的敏感性在细胞周期中变化很大， 激素只有在G1和S期晚期才有效（DNA 合成）阶段。 我们的长期目标是阐明 这种变化。 在本提案中，中心目标是测试 糖皮质激素受体（GR）磷酸化变化的假设 在细胞周期中，至少部分地解释了 灵敏度 这一假设基于GR数量依赖于细胞周期的证据， 核结合和磷酸化的研究已经得到了支持， 为了测试它--我们发现了一个快速的，依赖于糖皮质激素的增加， 磷酸化，我们通过磷酸肽图谱鉴定， 小鼠GR中7个磷酸化位点的测序：4个位于 控制细胞周期的p34 cdc 2激酶的共有序列。 该提案旨在回答以下具体问题： 1. 激素依赖性GR过度磷酸化和/或GR磷酸化 在细胞周期中的位置会发生变化吗 (a)同步化的WCL 2细胞（CHO 具有过表达的小鼠GR的细胞）将用32 P标记并处理 与激素一起测定GR的糖基化依赖性磷酸化;（B） 这些GR中的磷酸化位点将通过磷酸化肽鉴定。 绘制和测序任何新的肽。 2. 糖皮质激素敏感性和GR数量在细胞内是如何变化的 Cho细胞的周期 具有过表达GR的同步化CHO细胞将被 用于：（a）测量激素对稳定转染的细胞活性的影响， （B）确定GR基因和胸苷激酶的数量增加是否 G1/S中的结合位点是由于每个细胞增加的GR蛋白。 3. GR磷酸化突变体的行为与野生型GR不同吗？ 在正常磷酸化位点突变过表达GR的CHO细胞 将按照（2A）进行研究。 4. GR磷酸化和糖皮质激素诱导的细胞凋亡是否与糖尿病相关？ 淋巴细胞周期依赖性，可通过化疗改变 探员？ 同步WEHI-7细胞将用于测量（a）GR 如在la中的磷酸化;（B）糖皮质激素诱导的细胞凋亡;（c） 细胞周期调节剂在（a）和（B）中的作用。 我们的研究结果可能有助于治疗和提高对糖皮质激素的认识 阻力